Ibright cl1000

To determine plasma cytokine expression patterns upon radio(chemo)therapeutic treatment of HNSCC patients, cytokine arrays were performed. Supernatants from cell cultures were collected after incubation and instantly frozen with liquid nitrogen and preserved at -80 °C. Proteome Profiler^™ Human XL cytokine arrays (R&D Systems, Minneapolis, MN, USA) were hybridized with the cell culture medium as recommended by the supplier. Expression was visualized using an enhanced chemiluminescence detection kit (R&D Systems, Minneapolis, United States). Semiquantitative analysis was performed by measuring the density of the bands using an iBright CL 1000 biomolecular imager (Invitrogen, Carlsbad, CA, USA).
Plasma concentrations of chemokine CXCL11 were assessed from citrate-plasma samples and were determined by enzyme-linked immunosorbent assays (ELISA) according to manufacturer’s protocols (R&D Systems, Minneapolis, MN, USA).

The extracts of MC3TE-E1 for western blotting were prepared after the indicated treatment for 3 days as previously described [34 (link)]. About 20 μg protein from cells culture lysates were harvested using PRO-PREPTM protein extraction solution (Boca Scientific Inc., Boca Raton, FL) and carried out subsequent electrophoresis, according to the manufacturer’s instruction. The primary antibodies against the following proteins: FoxO1 (Abcam, 1:1000), SIRT1 (Abcam, 1:1000), superoxide dismutase 2 (SOD2, Abcam, 1:1000), Runx-2 (Abcam, 1:1000), OC (Abcam, 1:1000), and Cola1a (Abcam, 1:1000). Expression levels of the target protein were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Boster, Wuhan, China, 1:2000) levels in each sample. The following day, HRP-conjugated goat anti-rabbit, which was used as secondary antibodies, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and the results were detection and analysis by using an iBrightCL1000 (Invitrogen, Carlsbad, CA).

Whole brain tissue was homogenized by mixing with ProtinEx Animal Cell/Tissue (Gene All Biotechnology, Korea) containing 1% protease inhibitor followed by centrifugation at 13,000 ×g for 10 min at 4°C. Then, the sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated protein was transferred to a PVDF membrane (Millipore). To block the membrane, it was treated with 5% skim milk for 1 h, and then washed three times for 10 min using 1X Tris-buffered saline with 0.1% Tween® 20 (TBST) buffer. The membrane and primary antibody (1:1,000) solution were incubated for 12 h at 4°C. After incubation, secondary antibody (1:2,500) was reacted with the membrane for 1 h at room temperature, and the chemiluminescence band was detected using an image analyzer (iBright CL1000, Invitrogen, USA). Finally, the density of the band was calculated using ImageJ software (National Institutes of Health, USA).

Eosinophils were primed with cytokines for 20 h and were seeded on HA-IgG for the indicated times. Treatment with bafilomycin-A1 (BioViotaca, Liestal, Switzerland) started after priming and 20 min before seeding on IgG. Cells were lysed directly in LaemmLi buffer (10% SDS plus ß-mercaptoethanol), before boiling and loading onto 15 to 10% SDS-polyacrylamide gels. Proteins were transferred into a PVDF membrane. Immunoblotting was performed as previously described [22 (link)]. Blots were subsequently incubated with desired primary antibodies: anti-ß-actin from Sigma-Aldrich, anti-phospho-cofilin, anti-SQSTM1 and anti-LC3B from Cell Signaling Technology (Danvers, MA, USA) and anti-phospho-p38 from Genetel Laboratories (Pasadena, TX, USA) (all diluted by 2000-fold in 1× TBS, 0.1% Tween-20 with 5% BSA). Then, the appropriate HRP-conjugated secondary antibodies (Calbiochem, San Diego, CA, USA) were utilized. Immunoreactive bands were visualized using ECL reagents and GE LAS4000 chemiluminescence imager (GE Healthcare, Chicago, IL, USA) and iBright CL1000 (InVitrogen, ThermoFisher Scientifics, Waltham, MA, USA). Bands were quantified using ImageJ (https://imagej.nih.gov/ij/ accessed on 23 April 2018).

Gels with AnTat1.1 dtTomato cells were visualised by the fluorescence documentation system iBright CL1000 (Invitrogen). Exposure time was 100 ms. Images were combined into TIF-stacks with Fiji, and the colonies were aligned using the Plugin “Linear Stack Alignment with SIFT”. The projection expansion speeds were measured by manually drawing the tangents normal to the longitudinal centre line of each projection tip in successive frames and measuring the distances in Fiji.
Microscopy was performed with a fluorescence stereomicroscope (MZ16 FA, Leica), equipped with a 5x zoom objective and a CCD camera (pco.1600, pco), and with an inverted fluorescence microscope (DMI6000B, Leica), equipped with an automated stage (Pecon) and an incubation chamber (Leica). Sixty-millimetre and 35-mm gel dishes were fixed in the appropriate dish holders and incubated at $27{}^{\circ }\text{C}$ and 5 % ${\text{CO}}_2$ . Single projection tips were recorded at various time points by using a 20x, NA 0,4 phase contrast objective, in combined fluorescence and phase contrast mode (FLUO-PH, LASX software, 5 ms exposure time, minimal transmitted light). Recordings were 15 min each with a temporal resolution of 250 ms (4 fps).

Western blotting was performed using a gel documentation system (iBrightCL1000, Invitrogen and Image Lab Software version 3.0), and a standard protocol as previously described (Zhang et al., 2019 (link)). The primary antibodies were anti-cyclin dependent kinase inhibitor 2A (P16) (80772, Cell Signaling Technology, United States), anti-cyclin dependent kinase inhibitor 1A (P21) (2947, Cell Signaling Technology), P53 (21083, Signalway Antibody, United States), PTEN (ab31392, abcam, United Kingdom), anti-phosphorylated-AKT (p-AKT) (4060, Cell Signaling Technology) and anti-AKT (4691, Cell Signaling Technology); Cleaved caspase-3 (29034, Signalway Antibody), Bcl-2 (ab196495, abcam), vascular endothelial growth factor (VEGF, ab52917, abcam), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174, Cell Signaling Technology); TSG101 (14497, Proteintech, United States), CD63 (25682, Proteintech), CD81 (66866, Proteintech) and horseradish peroxidase-conjugated were secondary antibodies (Biosharp, China).

Whole-cell extracts were prepared by direct lysis with 1x loading buffer (Roti-Load^®, Roth) heated to 95 °C. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham) by western blotting, blocked in 5% BSA dissolved in TBS-Tween and incubated with specific primary antibodies and secondary peroxidase conjugated antibodies (Table A2). Detection was performed using the iBright CL1000 (Invitrogen) system.